Severe sepsis, defined as acute onset organ failure in the setting of infection, is the leading cause of death in noncoronary intensive care units in the US and costs billions of dollars per year.1
Sepsis is characterized by the overproduction of several different types of inflammatory mediators, which has been identified as one possible cause of the systemic inflammation and organ dysfunction seen in septic patients.2,3
An increase in the understanding of the immune response in sepsis4,5
has led to expanded research into blood purification therapies intended to reduce the concentration of circulating inflammatory mediators.6,10
The usefulness of blood purification, either filtration or adsorption of blood or plasma proteins, as a treatment for sepsis lies in its ability to remove a broad range of molecules rather than targeting single mediators.
Our group is currently developing a hemoadsorption device to treat severe sepsis by depleting circulating levels of pro- and anti-inflammatory cytokines. This cytokine adsorption device (CAD) consists of a column packed with biocompatible CytoSorb™
polymer beads (CytoSorbents, NJ), whose effectiveness for cytokine removal has been demonstrated in both in vitro
and ex vivo
We have modeled the performance of the beads and the device based on data obtained during scaled-down cytokine capture experiments for the cytokines interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-10 (IL-10). Our results indicate that the beads remove almost 90% of middle-molecular weight proteins such as IL-6 and IL-10 (18–21 kDa) after 4 h, but only 50–60% of the relatively large TNF trimer (52 kDa).13
This result is not surprising due to the limited range of pore sizes available on the surface of the CytoSorb™
beads: the pores are designed to exclude larger molecules such as albumin (66 kDa) and fibrinogen (340 kDa). Lixelle (Kaneka Corporation, Osaka, Japan), an alternative adsorbent material being tested to treat hypercytokinemia, has shown only 20% removal of TNF in 4 h using the same experimental setup as with the CytoSorb™
beads (unpublished data). Our conclusion is that beads which target cytokines nonspecifically are not capable of removing TNF at comparable levels to smaller cytokines while maintaining their ability to exclude larger proteins.
Increasing removal of TNF within our device is of particular interest, as sustained high concentrations of TNF are negatively correlated with survival in septic patients.14
Neutralization of TNF in small animal sepsis models using soluble receptors and monoclonal antibodies has been shown to reduce mortality15,16
and several candidates from each category of TNF-specific antagonists have been tested in clinical trials since 1993. A review of these trials demonstrates that no statistically significant improvement in patient mortality has been observed; in some cases, survival rates were actually significantly better in the placebo group.17
Many argue that these therapies have failed because they make no distinction between patients requiring immune suppression and those requiring immune augmentation, due to issues such as type of infection, timing, and severity of insult.18,19
Our approach currently provides for either type of immunomodulation for smaller proinflammatory and anti-inflammatory cytokines. We hypothesized that a combined approach of specific and non-specific cytokine capture would selectively increase capture of TNF to levels comparable to those of other cytokines, thus further increasing the efficacy of our device.
The main goal of this study was to accelerate the rate of removal and overall capacity for TNF capture by immobilizing anti-TNF on the outer surface of the beads in the CAD. We explored covalent versus passive immobilization techniques as well as several surface functional group amplification methods, including poly-L-lysine (PLL) cross-linking. We have also developed a simple method of bound antibody quantification which dramatically decreased the amount of time and resources involved in quantifying antibody binding for the various immobilization schemes. Passive adsorption of anti-TNF led to a 29% increase in TNF removal over covalent binding and a 43% increase over unmodified CytoSorb™ beads. Lastly, we characterized the retention of the passively adsorbed antibodies to suggest the clinical safety of treatment with a CAD containing adsorbed anti-TNF beads.